Epithelial cell scattering recapitulates the first steps of carcinoma invasion/metastasis. While the balance between cell-cell adhesive activity and cell motility ultimately determines this process, its molecular mechanisms remain unclear. Adherence junctions and tight junctions (TJs) are primarily responsible for cell-cell adhesive activity and subjected to dynamic remodeling. We previously showed that Rab13 and its effector protein JRAB/MICAL-L2 mediate the endocytic recycling of the integral TJ protein occludin and the assembly of functional TJs. In this study, we examined the role of Rab13 and JRAB/MICAL-L2 in the scattering of Madin-Darby canine kidney (MDCK) cells in response to 12-O-tetradecanoylphorbol-13-acetate (TPA). Knockdown of Rab13 in canine MDCK cells suppressed the TPA-induced scattering, and this phenotype was restored by re-expression of human Rab13. During TPA-induced MDCK cell scattering, Rab13 was transiently activated and returned to its basal level, and both Rab13 and JRAB/MICAL-L2 were colocalized with F-actin at cell-cell contact sites and then accumulated at emerging lamellipodial structures. TPA-induced MDCK cell scattering was also inhibited by knockdown of canine JRAB/MICAL-L2 and rescued by re-expression of mouse JRAB/MICAL-L2. These results indicate that Rab13 and JRAB/MICAL-L2 are involved in epithelial cell scattering.